Telomeres, the ends of eukaryotic chromosomes, are essential for genome integrity. They protect chromosomes from degradation and fusions and serve as specialized sites for gene expression. Cells distinguish telomeres from double strand breaks even though, paradoxically, the two structures have much in common. Because the conventional replication apparatus cannot duplicate the very ends of linear molecules, in most eukaryotes, telomeric DNA is maintained by a specialized reverse transcriptase, telomerase. The long term goal of this grant is to elucidate mechanisms that enable telomeres to fulfill their varied functions, using S. cerevisiae, and to a lesser extent, S. pombe as models. Experiments in this funding period will focus on the molecular basis of both cell cycle and telomere length regulation of telomerase. The first aim uses a combination of in vivo and in vitro approaches to determine requirements for stable telomerase assembly and activity on DNA ends. Specifically, we will test if long single-strand TG1-3 tails are required to form a stable holoenzyme-telomere complex and if certain mutations in telomere binding proteins and/or telomerase components are defective in this step. To determine the precise roles of Est1 and Est3, two telomerase accessory proteins that are essential in vivo but not in vitro, we will develop more physiological in vitro assays to determine if the two cooperate to overcome Cdc13 inhibition of telomerase elongation. Aim 2 focuses on Rif1, a telomere binding protein that inhibits telomerase activity in cis. We will test two hypotheses to explain why loss of Rif1 results in telomere hyper-elongation. The first is that Rif1 inhibits telomerase binding to telomeres, and this inhibition is relieved by its post translation modification once telomeres become short enough to be preferred substrates for telomerase. Biochemical and genetic approaches are proposed to identify these modifications. These studies will generate a set of rif1 alleles that will be useful for future functional studies on Ri1, regardless of its mode of action. The second model proposes that Rif1 promotes replication fork progression through telomeric DNA, and in its absence, forks stall and often break, and these breaks are lengthened by telomerase. Replication fork progression will be monitored by measuring DNA polymerase occupancy and by visualizing replication intermediates with two dimensional gels of telomeric DNA in Rif1-depleted cells. If increased pausing is detected in the absence of Rif1, a genetic assay will determine if this pausing is associated with breakage and telomerase-mediated elongation. Aim3 describes the continuation of a proteomics approach that uses mass spectrometry (MS) to identify novel telomerase interacting proteins and modifications of these proteins. By simultaneous over-expression and epitope tagging of multiple telomerase subunits, we isolated active telomerase in both S. cerevisiae and S. pombe and used MS to identify multiple associated proteins, several of which are known to affect telomere length. These new proteins and protein modifications will be studied by a combination of genetics and biochemistry to determine their exact roles in the telomerase process.